Suppressed Platelet Activation Research Articles

NADPH oxidase 1/4 dual inhibitor setanaxib suppresses platelet activation and thrombus formation

AimsThe production of reactive oxygen species (ROS) by NADPH oxidase (NOX) is able to induce platelet activation, making NOX a promising target for antiplatelet therapy. In this study, we examined the effects of setanaxib, a dual NOX1/4 inhibitor, on human platelet function and ROS-related signaling pathways. Materials and methodsIn collagen-stimulated human platelets, aggregometry, assessment of ROS and Ca2+, immunoblotting, ELISA, flow cytometery, platelet adhesion assay, and assessment of mouse arterial thrombosis were performed in this study. Key findingsSetanaxib inhibited both intracellular and extracellular ROS production in collagen-activated platelets. Additionally, setanaxib significantly inhibited collagen-induced platelet aggregation, P-selectin exposure from α-granule release, and ATP release from dense granules. Setanaxib blocked the specific tyrosine phosphorylation-mediated activation of Syk, LAT, Vav1, and Btk within collagen receptor signaling pathways, leading to reduced activation of PLCγ2, PKC, and Ca2+ mobilization. Setanaxib also inhibited collagen-induced activation of integrin αIIbβ3, which is linked to increased cGMP levels and VASP phosphorylation. Furthermore, setanaxib suppressed collagen-induced p38 MAPK activation, resulting in decreased phosphorylation of cytosolic PLA2 and reduced TXA2 generation. Setanaxib also inhibited ERK5 activation, affecting the exposure of procoagulant phosphatidylserine. Setanaxib reduced thrombus formation under shear conditions by preventing platelet adhesion to collagen. Finally, in vivo administration of setanaxib in animal models led to the inhibition of arterial thrombosis. SignificanceThis study is the first to show that setanaxib suppresses ROS generation, platelet activation, and collagen-induced thrombus formation, suggesting its potential use in treating thrombotic or cardiovascular diseases.

Eugenol: A Potential Modulator of Human Platelet Activation and Mouse Mesenteric Vascular Thrombosis via an Innovative cPLA2-NF-κB Signaling Axis.

Platelets, a type of anucleated cell, play a crucial role in cardiovascular diseases (CVDs). Therefore, targeting platelet activation is essential for mitigating CVDs. Endogenous agonists, such as collagen, activate platelets by initiating signal transduction through specific platelet receptors, leading to platelet aggregation. Eugenol, primarily sourced from clove oil, is known for its antibacterial, anticancer, and anti-inflammatory properties, making it a valuable medicinal agent. In our previous study, eugenol was shown to inhibit platelet aggregation induced by collagen and arachidonic acid. We concluded that eugenol exerts a potent inhibitory effect on platelet activation by targeting the PLCγ2-PKC and cPLA2-TxA2 pathways, thereby suppressing platelet aggregation. In our current study, we found that eugenol significantly inhibits NF-κB activation. This led us to investigate the relationship between the NF-κB and cPLA2 pathways to elucidate how eugenol suppresses platelet activation. In this study, we prepared platelet suspensions from the blood of healthy human donors to evaluate the inhibitory mechanisms of eugenol on platelet activation. We utilized immunoblotting and confocal microscopy to analyze these mechanisms in detail. Additionally, we assessed the anti-thrombotic effect of eugenol by observing fluorescein-induced platelet plug formation in the mesenteric microvessels of mice. For immunoblotting and confocal microscopy studies, eugenol significantly inhibited NF-κB-mediated signaling events stimulated by collagen in human platelets. Specifically, it reduced the phosphorylation of IKK and p65 and prevented the degradation of IκBα. Additionally, CAY10502, a cPLA2 inhibitor, significantly reduced NF-κB-mediated signaling events. In contrast, BAY11-7082, an IKK inhibitor, did not affect collagen-stimulated cPLA2 phosphorylation. These findings suggest that cPLA2 acts as an upstream regulator of NF-κB activation during platelet activation. Furthermore, both BAY11-7082 and CAY10502 significantly reduced the collagen-induced rise in intracellular calcium levels. In the animal study, eugenol demonstrated potential as an anti-thrombotic agent by significantly reducing platelet plug formation in fluorescein-irradiated mouse mesenteric microvessels. Our study uncovered a novel pathway in platelet activation involving the cPLA2-NF-κB axis, which plays a key role in the antiplatelet effects of eugenol. These findings suggest that eugenol could serve as a valuable and potent prophylactic or therapeutic option for arterial thrombosis.

Mechanism of Bile Acid in Regulating Platelet Function and Thrombotic Diseases.

Platelets play a key role in physiological hemostasis and pathological thrombosis. Based on the limitations of current antiplatelet drugs, it's important to elucidate the mechanisms of regulating platelet activation. In addition to dissolving lipid nutrients, bile acids (BAs) can regulate platelet function. However, the specific mechanisms underlying BAs-mediated effects on platelet activation and thrombotic diseases remain unknown. Therefore, the effects of BAs on platelets and intracellular regulatory mechanisms are explored. It is showed that the inhibitory effect of secondary BAs is more significant than that of primary BAs; lithocholic acid (LCA) shows the highest inhibitory effect. In the process of platelet activation, BAs suppress platelet activation via the spleen tyrosine kinase (SYK), protein kinase B (Akt), and extracellular signal-regulated kinase1/2 (Erk1/2) pathways. Nck adaptor proteins (NCK1) deficiency significantly suppress the activity of platelets and arterial thrombosis. Phosphorylated proteomics reveal that LCA inhibited phosphorylation of syntaxin-11 at S80/81 in platelets. Additional LCA supplementation attenuated atherosclerotic plaque development and reduced the inflammation in mice. In conclusion, BAs play key roles in platelet activation via Syk, Akt, ERK1/2, and syntaxin-11 pathways, which are associated with NCK1. The anti-platelet effects of BAs provide a theoretical basis for the prevention and therapy of thrombotic diseases.

The Effect of Individual Compounds from <i>Rubus chamaemoruson</i> Hemostasis <i>in vitro</i>

Introduction. One of the key tasks of the pharmaceutical industry is the search for new promising compounds – potential drug candidates. Natural objects, especially plants, have long been rich sources of new molecules and are widely used in the global food and pharmaceutical industries. Cloudberry (Rubus chamaemorus L.) is a perennial herb from the Rosaceae family. The fruits and leaves of R. chamaemorus contain a wide variety of polyphenolic secondary metabolites – hydrolysable/condensed tannins and flavonoids. Extracts enriched by polyphenols showed significant antiproliferative activity and inhibition of cell growth, and also induce cell apoptosis. As a result of our previous phytochemical research of R. chamaemorus leaves, five polyphenolic secondary metabolites belonging to the classes of tannins and flavonoids were isolated and characterized.Aim. Screening of previously isolated from R. chamaemorus individual compounds for the hemostasis system in vitro and identification of the most promising compounds for subsequent pharmaceutical development.Materials and methods. Experiments under in vitro conditions were performed on the blood of healthy male donors. The research of the effect on platelet aggregation was carried out according to the Born method on an AT-02 aggregometer (LLC "SPF "Medtech", Russia). Determination of anticoagulant activity was carried out by conventional clotting tests on a Solar CGL 2110 turbidimetric hemocoagulometer (CJSC "SOLAR", Russia). Cytofluorimetric analysis was performed on a NovoCyte instrument (Agilent Technologies, USA).Result and discussion. The influence of the isolated compounds 1–5 on the parameters of activation, platelet aggregation and the coagulation component of hemostasis was studied. At a concentration of 1.0 mg/ml, compounds 1–5 did not affect the fibrinogen concentration and prothrombin time. Compounds 1, 3 and 5 completely suppressed platelet activation at the studied concentrations. Compounds 1 and 3 showed antiaggregation activity comparible to the values of acetylsalicylic acid and are contained in all aqueous and alcoholic extracts of R. сhamaemorus leaves; their quantitative content varies depending on the extraction conditions.Conclusion. Thus, as a result of the screening of individual compounds 1–5 isolated from the leaves of R. chamaemorus their antiaggregating and anticoagulation properties were established. Compounds 1 (4-O-α-L-arabinofuranosylellagic acid) and 3 (quercetin-3-O-β-D-glucuronide) showed antiaggregation activity comparible to that of acetylsalicylic acid, and are the most promising of the studied series of compounds for the subsequent pharmaceutical development of new antiplatelet agents.

Eugenol Suppresses Platelet Activation and Mitigates Pulmonary Thromboembolism in Humans and Murine Models.

Platelets assume a pivotal role in the pathogenesis of cardiovascular diseases (CVDs), emphasizing their significance in disease progression. Consequently, addressing CVDs necessitates a targeted approach focused on mitigating platelet activation. Eugenol, predominantly derived from clove oil, is recognized for its antibacterial, anticancer, and anti-inflammatory properties, rendering it a valuable medicinal agent. This investigation delves into the intricate mechanisms through which eugenol influences human platelets. At a low concentration of 2 μM, eugenol demonstrates inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Notably, thrombin and U46619 remain unaffected by eugenol. Its modulatory effects extend to ATP release, P-selectin expression, and intracellular calcium levels ([Ca2+]i). Eugenol significantly inhibits various signaling cascades, including phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3β, mitogen-activated protein kinases, and cytosolic phospholipase A2 (cPLA2)/thromboxane A2 (TxA2) formation induced by collagen. Eugenol selectively inhibited cPLA2/TxA2 phosphorylation induced by AA, not affecting p38 MAPK. In ADP-treated mice, eugenol reduced occluded lung vessels by platelet thrombi without extending bleeding time. In conclusion, eugenol exerts a potent inhibitory effect on platelet activation, achieved through the inhibition of the PLCγ2-PKC and cPLA2-TxA2 cascade, consequently suppressing platelet aggregation. These findings underscore the potential therapeutic applications of eugenol in CVDs.

Blocking tumor-platelet crosstalk to prevent tumor metastasis via reprograming glycolysis using biomimetic membrane-hybridized liposomes

Activated platelets promote tumor progression and metastasis through active interactions with cancer cells, especially in promoting epithelial-mesenchymal transition (EMT) of tumor cells and shedding tumor cells into the blood. Blocking platelet-tumor cell interactions can be a potential strategy to inhibit tumor metastasis. Platelet activation requires energy produced from aerobic glycolysis. Based on this, we propose a platelet suppression strategy by reprogramming glucose metabolism of platelets, which has an advantage over conventional antiplatelet treatment that has a risk of serious hemorrhage. We develop a biomimetic delivery system using platelet membrane-hybridized liposomes (PM-Lipo) for codelivery of quercetin and shikonin to simultaneously inhibit lactate transporter MCT-4 and a glycolytic enzyme PKM2 for achieving metabolic reprogramming of platelets and suppressing platelet activation. Notably, PM-Lipo can also inhibit glycolysis in cancer cells, which actually takes “two-birds-one-stone” action. Consequently, the platelet-tumor cell interactions are inhibited. Moreover, PM-Lipo can bind with circulating tumor cells and reduce their seeding in the premetastatic microenvironment. The in vivo studies further demonstrated that PM-Lipo can effectively suppress primary tumor growth and reduce lung metastasis without affecting inherited functions of platelets. Reprogramming glycolysis of platelets can remodel the tumor immune microenvironment, including suppression of Treg and stimulation of CTLs.

Therapy with ticagrelor/prasugrel is associated with enhanced fibrinolysis and suppressed platelet activation as compared to clopidogrel in chronic coronary syndrome.

Therapy with ticagrelor/prasugrel is associated with enhanced fibrinolysis and suppressed platelet activation as compared to clopidogrel in chronic coronary syndrome.

Effect of evolocumab on platelet function in patients with acute coronary syndromes. An analysis of the randomized, double-blind, placebo-controlled EVOPACS Trial

Abstract Background Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors substantially lower levels of low-density lipoprotein cholesterol (LDL-C) and favourably impact clinical outcomes in patients with acute coronary syndromes (ACS). Although PCSK9 inhibitors have been suggested in ex vivo studies to suppress platelet activation, the impact of evolocumab on platelet function in ACS patients receiving potent antiplatelet therapy remains largely unknown. Methods The EVOPACS trial was a randomized, double-blind, placebo-controlled trial assessing the use of evolocumab initiated in the acute phase of ACS. Patients presenting with ST-elevation myocardial infarction (STEMI) or Non-ST-elevation ACS (NSTE-ACS) were randomly allocated in a 1:1 ratio to either evolocumab 420mg SC or matching placebo at baseline (during hospitalization for index ACS event) and after 4 weeks. All patients received atorvastatin 40mg daily. Dual antiplatelet therapy consisting of aspirin and a potent P2Y12 inhibitor was initiated according to STEMI / NSTE-ACS guidelines, unless a less potent treatment was indicated (e.g. due to concomitant use of oral anticoagulation). Platelet function was measured at baseline (during index hospitalization) and after 8 weeks, using the Multiplate analyser. The main outcome for this analysis was the change from baseline to 8 weeks in adenosine diphosphate (ADP)-induced platelet aggregation. Results Serial platelet function measurements were available in 260 of all 308 enrolled patients, 124 in the evolocumab and 136 in the placebo group. Mean age was 60.2±11.0 years and 83% were men. Baseline clinical characteristics were well balanced between the two treatment groups. At discharge, the proportion of patients treated with ticagrelor, prasugrel, and clopidogrel was 73%, 11%, and 9% respectively, without differences between groups. Evolocumab significantly reduced LDL-C compared to placebo at 8 weeks (difference in mean percentage change -40.75%, 95% CI -45.39 to -36.11; p<0.001). Change in ADP-induced platelet aggregation between baseline and 8-week follow-up was -5.6±23.5 U (from 24.1±19.8 to 29.7±25.6 U) in the evolocumab group vs. -1.7±198.7 U (from 23.0±14.7 U to 24.7±19.1 U) in the placebo group (p=0.13). Similar results were found in a subgroup of 189 patients (86 in the evolocumab and 103 in the placebo group) treated with ticagrelor at discharge, showing a change in ADP-induced platelet aggregation of -2.7±19.5 U with evolocumab vs. -2.4±19.9 U with placebo (p=0.80). Conclusions In this randomized trial of ACS patients largely treated with a potent P2Y12 inhibitor, evolocumab initiated during index hospitalization on top of high-intensity statin therapy had no measurable effect on ADP-induced platelet aggregation. These findings suggest that the early therapeutic benefit of PCSK9 inhibitors in ACS patients is likely related to plaque regression/stabilization rather than changes in platelet activity.

Abstract 131: Identification Of A Molecule To Modulate A Platelet Olfactory Receptor And Limit Thrombosis

Background and objective: Olfactory Receptors (ORs) are G protein-coupled receptors (GPCR) we recently discovered in human platelets. OR2L13 activation by an olfactory ligand Levo-Carvone (L-Carvone) limits platelet activation. Our goal was to better characterize the pharmacology of OR2L13 ligands through structural modifications of L-Carvone and by using an 8K bioactive non-olfactory ligand library and deciphering their role as anti-thrombotic. Methods and Results: With an IC 50 of 58 μM, 2-tert-Butyl-5-Methylphenol was the best odorant for inhibiting ADP-mediated platelet activation (Fig. 1). 169 molecular hits >25% threshold in an OR2L13-expressiong reporter cell line for cyclic AMP (cAMP) production were found. By counter screen (cell line without OR2L13 receptor), 12 ligands were identified for OR2L13 and 6 (CCF0054500, CCF0054432, CCF0053070, CCF0052249, CCF0051970, CCF0058399) made endogenous cAMP. Healthy platelet rich plasma (PRP) (n=7) was incubated with OR2L13 ligand for 30 minutes, then subjected to Light Transmission Aggregometry (LTA). 3/6 ligands (CCF0054500, CCF0052249 and CCF0058399) inhibited platelet aggregation through the P2Y 12 receptor and so were further validated by FACS using washed healthy platelets (n=8, attenuation of agonist-activated platelets surface P-selectin expression) for the following receptors: ADP, TRAP-6, U46619, and CRP. CCF0054500 suppressed platelet activation and aggregation through P2Y 12, PAR-1, Thromboxane and GPVI receptors. Whole blood (n=5) traversing collagen-coated capillaries reduced thrombosis when compared to vehicle under arterial sheer stress with CCF0054500. Conclusions: Like neurons, platelets have a fully functional OR signal transduction cascade. Platelet OR2L13 operates as a negative regulator of platelet reactivity and thrombosis in response to odorant and non-odorant surface agonists (biochemical activation) and under shear stress (biomechanical activation).

STING activation in platelets aggravates septic thrombosis by enhancing platelet activation and granule secretion

Sepsis is a dysregulated inflammatory consequence of systemic infection. As a result, excessive platelet activation leads to thrombosis and coagulopathy, but we currently lack sufficient understanding of these processes. Here, using the cecal ligation and puncture (CLP) model of sepsis, we observed septic thrombosis and neutrophil extracellular trap formation (NETosis) within the mouse vasculature by intravital microscopy. STING activation in platelets was a critical driver of sepsis-induced pathology. Platelet-specific STING deficiency suppressed platelet activation and granule secretion, which alleviated sepsis-induced intravascular thrombosis and NETosis in mice. Mechanistically, sepsis-derived cGAMP promoted the binding of STING to STXBP2, the assembly of SNARE complex, granule secretion, and subsequent septic thrombosis, which probably depended on the palmitoylation of STING. We generated a peptide, C-ST5, to block STING binding to STXBP2. Septic mice treated with C-ST5 showed reduced thrombosis. Overall, platelet activation via STING reveals a potential strategy for limiting life-threatening sepsis-mediated coagulopathy.

Stimulation of platelet P2Y1 receptors by different endogenous nucleotides leads to functional selectivity via biased signalling.

Platelet function during inflammation is dependent on activation by endogenous nucleotides. Non-canonical signalling via the P2Y1 receptor is important for these non-thrombotic functions of platelets. However, apart from ADP, the role of other endogenous nucleotides acting as agonists at P2Y1 receptors is unknown. This study compared the effects of ADP, Ap3A, NAD+ , ADP-ribose, and Up4A on platelet functions contributing to inflammation or haemostasis. Platelets obtained from healthy human volunteers were incubated with ADP, Ap3A, NAD+ , ADP-ribose, or Up4A, with aggregation and fibrinogen binding measured (examples of function during haemostasis) or before exposure to fMLP to measure platelet chemotaxis (an inflammatory function). In silico molecular docking of these nucleotides to the binding pocket of P2Y1 receptors was then assessed. Platelet aggregation and binding to fibrinogen induced by ADP was not mimicked by NAD+ , ADP-ribose, and Up4A. However, these endogenous nucleotides induced P2Y1 -dependent platelet chemotaxis, an effect that required RhoA and Rac-1 activity, but not canonical PLC activity. Analysis of molecular docking of the P2Y1 receptor revealed distinct differences of amino acid interactions and depth of fit within the binding pocket for Ap3A, NAD+ , ADP-ribose, or Up4A compared with ADP. Platelet function (aggregation vs motility) can be differentially modulated by biased-agonist activation of P2Y1 receptors. This may be due to the character of the ligand-binding pocket interaction. This has implications for future therapeutic strategies aimed to suppress platelet activation during inflammation without affecting haemostasis as is the requirement of current ant-platelet drugs. This article is part of a themed issue on Platelet purinergic receptor and non-thrombotic disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.4/issuetoc.

Sodium ferulate-functionalized silver nanopyramides with synergistic antithrombotic activity for thromboprophylaxis

Thrombosis is one of the feared complications contributing to death in patients with cardiovascular diseases. Ferulic acid (FA), a bioactive compound extracted from traditional Chinese medicines, has drawn tremendous attention in prevention and treatment of thrombosis because of its notable antithrombotic potency. However, the poor aqueous solubility and pharmacokinetic profile of FA limit its clinical use. In this study, sodium ferulate-functionalized silver nanopyramides (SF-pAgNPs) with narrow size distribution were synthesized to overcome these obstacles and enhance the suppression effect of platelet activation and thrombosis formation. The cytotoxicity and hemolysis assays demonstrated that the prepared nanopyramides have a favorable biocompatibility pattern. In vitro studies revealed that SF-pAgNPs could effectively suppress platelet activation, aggregation and adhesion through the synergetic antithrombotic potential of SF and nano silver. Furthermore, SF-pAgNPs exhibited potent antithrombotic activity and prolonged inhibitory effect, much better than PEG-Ag and free SF in mouse model. With enhanced antithrombotic effect and acceptable biocompatibility, we believe the sodium ferulate-functionalized silver nanopyramides might hold the potential to be a promising strategy for the prevention and treatment of thrombosis.

HMGB1-activatied NLRP3 inflammasome induces thrombocytopenia in heatstroke rat.

BackgroundThrombocytopenia, an early common complication in heatstroke (HS), has been widely considered as a mortality predictor of HS. The mechanism underlying thrombocytopenia in HS remains unknown. It is not known whether NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is activated in HS platelet, which, in turn, induces platelet activation and thrombocytopenia. This study tried to clarify the activation of the NOD-like receptor signaling pathway under HS conditions and investigate its roles in mediating HS-induced thrombocytopenia.MethodsRat HS models were established in a certain ambient temperature and humidity. Platelets, isolated from blood, were counted and CD62P, an index of platelet activation, was measured by flow cytometry in all rats. The colocalization of NLRP3 inflammasome in platelet was detected by confocal fluorescence microscopy. Mitochondrial-derived reactive oxygen species (ROS) was detected using the molecular probes. Plasma HMGB1 and IL-1β levels were measured by ELISA.ResultsPlatelet activation, showed by upregulated CD62P, and thrombocytopenia were observed in HS rats. HS activated the NLRP3 inflammasome, which was induced by elevated levels of ROS, while the upregulated CD62P and thrombocytopenia triggered by NLRP3 inflammasome were attributed to the high mobility group box protein 1 (HMGB1) inplasma. Moreover, inhibition of the NOD-like receptor signaling pathway in rats with HS suppressed platelet activation and the decline of platelet count. Similar results were obtained when the receptor toll-like receptor 4 (TLR4)/advanced glycation end product (RAGE) was blocked.ConclusionsThe NOD-like receptor signaling pathway induces platelet activation and thrombocytopenia in HS rats. These findings suggested that the NLRP3 inflammasome might be the potential target for HS treatment.

Stachydrine exhibits a novel antiplatelet property and ameliorates platelet-mediated thrombo-inflammation.

Platelets are versatile anucleate cells involved in thrombosis as well as inflammation. Stachydrine (STA), a major bioactive compound extracted from Motherwort, has multiple pharmacological properties. Nevertheless, the significance of STA in platelet regulation and whether STA could ameliorate platelet-mediated thrombo-inflammation still remain elusive. Human platelets were used to assess the regulatory effects of STA on platelet activation and interactions with neutrophils in vitro. FeCl3 injury-induced carotid/mesenteric thrombosis and collagen/epinephrine-induced pulmonary thromboembolism model were used to explore whether STA could regulate thrombosis in vivo. Furthermore, a cecal ligation and puncture-induced sepsis model was employed to investigate the role of STA in thrombo-inflammatory diseases. STA markedly suppressed platelet activation represented by aggregation, secretion, αIIbβ3-mediated signaling events and calcium mobilization, etc. by inhibiting agonists-induced activation signaling and potentiating cGMP-dependent inhibitory signaling. Mice receiving STA-treated platelets were less susceptible to thrombosis in vivo. In addition, decreased platelet-neutrophil interactions including platelet-neutrophil aggregates and neutrophil extracellular traps, and alleviative sepsis-induced multiorgan damage were observed due to STA-mediated platelet inhibition. This study suggested the potential therapeutic role of STA in thrombotic and thrombo-inflammatory disorders.

Dual surface modification of poly(L-lactide) scaffold achieved by thermal incorporation of aligned nanofiber and click immobilization of VEGF to enhance endothelialization and blood compatibility

Rapid endothelialization is very important in the development of blood-contact biomaterials because of the inhibitory effects of endothelium in preventing thrombosis and restenosis. In this study, topographical and biochemical cues were generated to accelerate endothelialization on the poly(L-lactide) (PLLA) scaffold via a simple, bio-orthogonal, and biocompatible process. Topographical guidance was incorporated into a PLLA scaffold via thermal attachment of aligned PLLA nanofibers using electrospinning. Vascular endothelial growth factor (VEGF) was covalently immobilized onto the PLLA scaffold by aminolysis and copper-catalyzed azide/alkyne cycloaddition-mediated click chemistry. Various surface analyses confirmed the presence of aligned topography and immobilized VEGF without delamination or leaching. In vitro experiments with human coronary artery endothelial cells revealed that proliferation, CD31 expression, and migration was significantly enhanced on the nanofiber-attached VEGF-immobilized PLLA scaffold. The aligned nanofiber also accommodated the sustained release of heparin, which suppressed the proliferation of human coronary artery smooth muscle cells and platelet activation without affecting surface endothelialization. The heparin-loaded, aligned, nanofiber-attached VEGF-immobilized PLLA scaffold was evidenced to be biocompatible and a promising candidate for blood-contact materials where fast endothelialization and suppressed platelet activation is required.

Targeting AnxA1/Formyl Peptide Receptor 2 Pathway Affords Protection against Pathological Thrombo-Inflammation.

Stroke is a leading cause of death and disability globally and is associated with a number of co-morbidities including sepsis and sickle cell disease (SCD). Despite thrombo-inflammation underlying these co-morbidities, its pathogenesis remains complicated and drug discovery programs aimed at reducing and resolving the detrimental effects remain a major therapeutic challenge. The objective of this study was to assess whether the anti-inflammatory pro-resolving protein Annexin A1 (AnxA1) was able to reduce inflammation-induced thrombosis and suppress platelet activation and thrombus formation in the cerebral microvasculature. Using two distinct models of pathological thrombo-inflammation (lipopolysaccharide (LPS) and sickle transgenic mice (STM)), thrombosis was induced in the murine brain using photoactivation (light/dye) coupled with intravital microscopy. The heightened inflammation-induced microvascular thrombosis present in these two distinct thrombo-inflammatory models was inhibited significantly by the administration of AnxA1 mimetic peptide AnxA1Ac2-26 (an effect more pronounced in the SCD model vs. the endotoxin model) and mediated by the key resolution receptor, Fpr2/ALX. Furthermore, AnxA1Ac2-26 treatment was able to hamper platelet aggregation by reducing platelet stimulation and aggregation (by moderating αIIbβ3 and P-selectin). These findings suggest that targeting the AnxA1/Fpr2/ALX pathway represents an attractive novel treatment strategy for resolving thrombo-inflammation, counteracting e.g., stroke in high-risk patient cohorts.

Novel potent antiplatelet thrombotic agent derived from biguanide for ischemic stroke

Platelet thrombosis is the main pathogeny resulting in the low curability of ischemic stroke, a leading cause of mortality and disability worldwide. Metformin, a biguanide derivative that is the first-line oral medicine for type 2 diabetes, alleviates the severity of ischemic stroke in diabetic patients and suppresses platelet activation in experimental animal model. However, the clinical implementation of commercial biguanide analogs for stroke related to platelet thrombosis remains challenging due to its weak potency, poor pharmacokinetic characteristics and possible hypoglycemia. Here, twenty-three biguanide derivatives were designed and synthesized based on the principles of bioisosteres. These derivatives were evaluated for the activity of antiplatelet thrombosis in vivo. We found that N-trifluoromethanesulfonyl biguanide derivative, compound b10, uniquely prevented cerebral infarction as well as neuronal function injury, and significantly decrease the mortality rate of ischemic stroke in the middle cerebral artery occlusion mice without significant side effects. We verified that b10 directly inhibited platelets thrombus formation and decreased the compactness of stroke thrombi. Particularly, b10 exhibited good potency to inhibit human platelet activation including platelet aggregation, adhesion, pseudopodia formation, integrin GPIIb/IIIa activation, CD62P expression and clot retraction. Meanwhile, the pharmacokinetics assessment showed that b10 had satisfying pharmacological characteristics including a longer duration and a higher oral absorption ratio than its parent compound. In addition, b10 remarkably ameliorated not only stroke related to platelet thrombosis but also carotid artery thrombus formation. It is concluded that the novel potent antiplatelet thrombotic agent derived from biguanide is a promising candidate for stroke treatment.

Role of Platelet Glycoprotein VI and Tyrosine Kinase Syk in Thrombus Formation on Collagen-Like Surfaces.

Platelet interaction with collagens, via von Willebrand factor, is a potent trigger of shear-dependent thrombus formation mediated by subsequent engagement of the signaling collagen receptor glycoprotein (GP)VI, enforced by integrin α2β1. Protein tyrosine kinase Syk is central in the GPVI-induced signaling pathway, leading to elevated cytosolic Ca2+. We aimed to determine the Syk-mediated thrombogenic activity of several collagen peptides and (fibrillar) type I and III collagens. High-shear perfusion of blood over microspots of these substances resulted in thrombus formation, which was assessed by eight parameters and was indicative of platelet adhesion, activation, aggregation, and contraction, which were affected by the Syk inhibitor PRT-060318. In platelet suspensions, only collagen peptides containing the consensus GPVI-activating sequence (GPO)n and Horm-type collagen evoked Syk-dependent Ca2+ rises. In whole blood under flow, Syk inhibition suppressed platelet activation and aggregation parameters for the collagen peptides with or without a (GPO)n sequence and for all of the collagens. Prediction models based on a regression analysis indicated a mixed role of GPVI in thrombus formation on fibrillar collagens, which was abolished by Syk inhibition. Together, these findings indicate that GPVI-dependent signaling through Syk supports platelet activation in thrombus formation on collagen-like structures regardless of the presence of a (GPO)n sequence.

Esculetin, a Coumarin Derivative, Prevents Thrombosis: Inhibitory Signaling on PLCγ2-PKC-AKT Activation in Human Platelets.

Esculetin, a bioactive 6,7-dihydroxy derivative of coumarin, possesses pharmacological activities against obesity, diabetes, renal failure, and cardiovascular disorders (CVDs). Platelet activation plays a major role in CVDs. Thus, disrupting platelet activation represents an attractive therapeutic target. We examined the effect of esculetin in human platelet activation and experimental mouse models. At 10–80 μM, esculetin inhibited collagen- and arachidonic acid-induced platelet aggregation in washed human platelets. However, it had no effects on other agonists such as thrombin and U46619. Esculetin inhibited adenosine triphosphate release, P-selectin expression, hydroxyl radical (OH·) formation, Akt activation, and phospholipase C (PLC)γ2/protein kinase C (PKC) phosphorylation, but did not diminish mitogen-activated protein kinase phosphorylation in collagen-activated human platelets. Platelet function analysis indicated that esculetin substantially prolonged the closure time of whole blood. In experimental mice, esculetin significantly increased the occlusion time in thrombotic platelet plug formation and reduced mortality associated with acute pulmonary thromboembolism. However, it did not prolong the bleeding time. This study demonstrates that esculetin inhibits human platelet activation via hindering the PLCγ2–PKC cascade, hydroxyl radical formation, Akt activation, and ultimately suppressing platelet activation. Therefore, esculetin may act as an essential therapeutic agent for preventing thromboembolic diseases.

Dual actions of group B Streptococcus capsular sialic acid provide resistance to platelet-mediated antimicrobial killing

Circulating platelets have important functions in thrombosis and in modulating immune and inflammatory responses. However, the role of platelets in innate immunity to bacterial infection is largely unexplored. While human platelets rapidly kill Staphylococcus aureus, we found the neonatal pathogen group B Streptococcus (GBS) to be remarkably resistant to platelet killing. GBS possesses a capsule polysaccharide (CPS) with terminal α2,3-linked sialic acid (Sia) residues that mimic a common epitope present on the human cell surface glycocalyx. A GBS mutant deficient in CPS Sia was more efficiently killed by human platelets, thrombin-activated platelet releasate, and synthetic platelet-associated antimicrobial peptides. GBS Sia is known to bind inhibitory Sia-recognizing Ig superfamily lectins (Siglecs) to block neutrophil and macrophage activation. We show that human platelets also express high levels of inhibitory Siglec-9 on their surface, and that GBS can engage this receptor in a Sia-dependent manner to suppress platelet activation. In a mouse i.v. infection model, antibody-mediated platelet depletion increased susceptibility to platelet-sensitive S. aureus but did not alter susceptibility to platelet-resistant GBS. Elimination of murine inhibitory Siglec-E partially reversed platelet suppression in response to GBS infection. We conclude that GBS Sia has dual roles in counteracting platelet antimicrobial immunity: conferring intrinsic resistance to platelet-derived antimicrobial components and inhibiting platelet activation through engagement of inhibitory Siglecs. We report a bacterial virulence factor for evasion of platelet-mediated innate immunity.